The vagus nerve mediates behavioural depression, but not fever, in response to peripheral immune...

The vagus nerve mediates behavioural depression, butnot fever, in response to peripheral immune signals; afunctional anatomical analysis

Jan Pieter Konsman,1 Giamal N. Luheshi,2 Rose-Marie BlutheÂ1 and Robert Dantzer1

1INSERM U394, Neurobiologie InteÂgrative, Institut FrancËois Magendie, Rue Camille Saint-SaeÈns, 33077 Bordeaux Cedex, France2Douglas Hospital Research Center, 6875, Boulevard LaSalle, Verdun, Quebec, Canada H4H 1R3

Keywords: Fos, interleukin-1, neuro±immune interactions, rat, Stat3

Abstract

Cytokines act on the brain to induce fever and behavioural depression after infection. Although several mechanisms of cytokine-

to-brain communication have been proposed, their physiological signi®cance is unclear. We propose that behavioural depression

is mediated by the vagus nerve activating limbic structures, while fever would primarily be due to humoral mechanisms affectingthe preoptic area, including interleukin-6 (IL-6) action on the organum vasculosum of the laminae terminalis (OVLT) and induction

of prostaglandins. This study assessed the effects of subdiaphragmatic vagotomy in rats on fever, behavioural depression, as

measured by the social interaction test, and Fos expression in the brain. These responses were compared with induction of the

prostaglandin-producing enzyme cyclooxygenase-2 and the transcription factor Stat3 that translocates after binding of IL-6.Vagotomy blocked behavioural depression after intraperitoneal injection of recombinant rat IL-1b (25 mg/kg) or lipopolysaccharide

(250 mg/kg; LPS) and prevented Fos expression in limbic structures and ventromedial preoptic area, but not in the OVLT. Fever

was not affected by vagotomy, but associated with translocation of Stat3 in the OVLT and cyclooxygenase-2 induction aroundblood vessels. These results indicate that the recently proposed vagal link between the immune system and the brain activates

limbic structures to induce behavioural depression after abdominal in¯ammation. Although the vagus might play a role in fever in

response to low doses of LPS by activating the ventromedial preoptic area, it is likely to be overridden during more severeinfection by action of circulating IL-6 on the OVLT or prostaglandins induced along blood vessels of the preoptic area.

Introduction

Detection of bacterial lipopolysaccharide (LPS) by immune cells

leads to interleukin-1b (IL-1b) and interleukin-6 (IL-6) release. These

cytokines act on the brain to provoke fever and behavioural

depression (Rothwell & Hopkins, 1995). Fever is an adaptive

response to infection (Kluger, 1991) and behavioural depression

contributes to it by reducing energy expenditure (Hart, 1988).

Because of their size and hydrophilic nature, cytokines cannot

diffuse across the blood±brain barrier (BBB). Three possible

mechanisms of cytokine-to-brain signalling have been proposed.

Classically, cytokines are thought to act on circumventricular organs

where the BBB is absent (Blatteis, 1992). IL-6, for example, rapidly

rises in plasma after LPS administration (Luheshi et al., 1996) and

induces the cellular activation marker c-fos exclusively in circum-

ventricular organs (VallieÁres et al., 1997). Alternatively, LPS and IL-

1b induce cyclooxygenase-2 (COX-2), the limiting enzyme for

prostaglandin synthesis, in endothelial cells (Cao et al., 1996;

La¯amme et al., 1999). Lipophilic prostaglandins freely diffuse

across the BBB and might thus act on receptors found on neuronal

populations (Zhang & Rivest, 1999). Recently, the vagus nerve was

proposed as an immune-to-brain pathway (Dantzer, 1994). Although

evidence exists for each of these mechanisms, their possible

intervention has usually been tested separately and by addressing

only one physiological response.

Transection of the vagus nerves inhibits fever only in response to

low doses of LPS or IL-1b (Hansen & Krueger, 1997; Romanovsky

et al., 1997). Clearly, higher amounts of circulating cytokines are

more prone to act on circumventricular organs or induce COX-2

around blood vessels. However, vagotomy consistently blocks LPS-

and IL-1b-induced behavioural depression, as measured by the social

interaction test, even in response to high doses (BlutheÂ et al., 1994,

1996b). Since higher doses of IL-1b are needed to induce behavioural

depression than fever (Anforth et al., 1998), it is possible that fever

and behavioural depression are mediated by distinct pathways of

immune-to-brain communication, resulting in activation of different

brain structures. In the case of fever, the preoptic hypothalamus is the

target structure (Boulant, 1981). The neural structures implicated in

behavioural changes are still unknown, but limbic structures are good

candidates.

This study was designed to test the hypothesis that fever and

behavioural depression are due to different mechanisms of immune-

to-brain signalling. In addition to fever and behavioural depression,

we evaluated LPS-induced Fos expression in rats that underwent

subdiaphragmatic vagotomy and compared it with COX-2 induction.

Brain targets of IL-6 were investigated by studying expression of

Stat3 that translocates into the nucleus after activation by the signal

Correspondence: Dr Jan Pieter Konsman, Neuroscience, Floor 11, Division ofCell Biology, Department of Biomedicine and Surgery, Faculty of HealthSciences, 58185 LinkoÈping, Sweden.E-mail: [email protected]

Received 4 February 2000, revised 7 September 2000, accepted 25 September2000

European Journal of Neuroscience, Vol. 12, pp. 4434±4446, 2000 ã Federation of European Neuroscience Societies

transducer gp130 common to the IL-6 receptor family (Hirano et al.,

1994; Heinrich et al., 1998).

The mechanisms behind the effects of vagotomy are still unclear.

Vagotomy may interrupt transmission of a peripheral IL-1b signal to

the nucleus of the solitary tract (NTS) where vagal ®bres terminate.

Alternatively, attenuated IL-1b induction in the brain of vagotomized

animals has been proposed to underlie its attenuating effects (LayeÂ

et al., 1995). Therefore, both LPS-induced Fos expression in the NTS

and IL-1b immunoreactivity in the brain were assessed.

Materials and methods

Animals

All experiments were performed on adult male Wistar rats (Charles

River, Saint Aubin les Elbeuf, France) of 225±250 g body weight.

The animals were housed in a temperature-controlled room

(21 6 2 °C) arti®cially lit between 08.00 h and 20.00 h, and were

provided with food (Extralabo, Provins, France) and water ad libitum.

Juvenile male rats (21±35 days of age) of the same strain served as

stimulus animals for the behavioural studies. All animal experiments

were conducted in accordance with French legislation.

Surgery

One hundred and sixteen animals were food deprived for 24 h prior to

surgery and randomly assigned to vagotomy or sham operations, as

previously described (BlutheÂ et al., 1996a). Brie¯y, animals were

anaesthetized with a mixture of ketamine and xylazine (61 and 9 mg/

kg, respectively; RhoÃne MeÂrieux, France and Bayer Pharma, Sens,

France), and the main ventral and dorsal trunks of the vagus nerve

were transected immediately above the stomach. All connective

tissue 2±3 cm between the gastric artery and the oesophagus was

removed to ensure the transection of accessory vagal branches. Sham

animals underwent the same surgical procedure excluding nerve

transection. All rats received a single intramuscular injection of

amoxilin (75 mg/kg) and were allowed to recover for 3 weeks.

During the ®rst week of recovery, animals were offered highly

palatable food, in addition to normal chow. Palatable food has

previously been shown to reduce body weight loss (Kraly et al., 1986)

and differences in basal body temperature in vagotomized animals

(Romanovsky et al., 1997). Thirty-®ve animals which continued to

lose weight or which showed apparent signs of sickness, such as

piloerection, were euthanized.

In the 34 animals assigned to fever and behavioural studies,

vagotomy was veri®ed by blockade of the anorexic effect of

intraperitoneal (i.p.) injection of cholecystokinin (4 mg/kg; Sigma,

St Fallavier, France) (Smith et al., 1981) 2 weeks after the end of the

studies. The rats were deprived of food for 18 h, then injected with

cholecystokinin or saline and presented with food 5 min later. Food

intake was measured over the following 30 min. In the 38 animals

assigned to immunocytochemical studies, vagotomy was veri®ed by

incubating NTS sections with isolectin I-B4 (1 mg/mL phosphate-

buffered saline; L5391, Sigma, St Fallavier, France) which was

previously shown to bind to vagal visceral afferents in the NTS (Li

et al., 1997).

Experimental protocol

Injections were administered i.p. (1.0 mL/kg) to hand-held, lightly

restrained rats. Rats were injected with either 25 mg/kg recombinant

rat IL-1b (rrIL-1b; speci®c activity 317 IU/mg; Dr S. Poole, NIBSC,

Potters Bar, UK) dissolved in pyrogen-free saline containing 0.1%

endotoxin-free bovine serum albumin (BSA; A-8806, Sigma) or

250 mg/kg of LPS (Escherichia coli O127:B8, Sigma) dissolved in

pyrogen-free saline. Control injections consisted of pyrogen-free

saline alone. These doses of rrIL-1b and LPS were previously shown

to reliably induce behavioural depression and robust fever responses

from which animals recover within 24 h (Luheshi, unpublished

observations) (BlutheÂ et al., 1992; Anforth et al., 1998; Konsman

et al., 1999).

Fever

Fever studies were performed at an ambient temperature of

21 6 2 °C, that is below the thermal neutrality for rats (28 °C).

Hypothermia can occur at ambient temperatures below thermal

neutrality when high doses (1000 mg/kg) of LPS are administrated

intravenously (Romanovsky et al., 1997), raising the possibility that

fever phases are masked. However, hypothermia does not occur at

lower doses of LPS (10 mg/kg intravenously) (Romanovsky et al.,

1998). Since i.p. administration of LPS results in 10±100-fold lower

circulating levels compared with intravenous administration (Yasui

et al., 1995), fever phases are very unlikely to be masked after i.p.

injection of 250 mg/kg LPS when monitoring body temperature at

21 °C. Core body temperature was measured continuously in

conscious, undisturbed, individually housed animals by remote

radio-telemetry, via battery-operated biotelemetry transmitters (Data

Sciences, St Paul, Minnesota, USA), previously implanted in the

abdominal cavity during vagotomy or sham surgery. The output

frequency (Hz) of each transmitter was monitored by an antenna

mounted in a receiver board situated beneath the cage of each animal,

and channelled to a peripheral processor (Dataquest IV, Data

Sciences). Frequencies were sampled at 10-min intervals and

converted to degrees Celsius (°C). Since the pyrogenic effects of

IL-1b and LPS are most pronounced during the light phase (Opp &

Toth, 1998), animals received a single injection of rrIL-1b, LPS or

vehicle at 10.00 h and their temperatures were measured every

10 min for 7 h. The mean of three consecutive measurements for

each individual animal was used for further analysis.

Behavioural depression

Following a 2-week recovery period from the fever study, social

interaction was tested in the same group of animals. In this

experiment, rats were injected with LPS, rrIL-1b or vehicle. All

animals received an injection of vehicle and the treatment groups

were arranged such that rats that had received LPS in the fever study

were injected with rrIL-1b, whereas those rats that had received rrIL-

1b previously were injected with LPS. This experimental design was

adopted to avoid the development of tolerance to LPS. Behavioural

depression was induced by injection of rrIL-1b, or LPS assessed by

reduction in the duration of social interaction with a conspeci®c

juvenile introduced into the home cage of the test animal for a 4-min

observation session, immediately before and 2 h after treatment. This

time-point was chosen because it corresponds to the appearance of

effects of IL-1b and LPS on behaviour (BlutheÂ et al., 1992). Social

interaction was measured by the duration of ano-genital snif®ng and

active interaction of the adult with the juvenile. Different juveniles

were presented on each occasion to avoid habituation. Durations of

social interaction measured in animals treated with saline were used

as baseline values. Social interaction studies were carried out during

the dark phase of the cycle, between 20:00 h and 01:00 h, since rats

display increased social behaviour during the dark phase.

Tissue processing

Rats assigned to immunocytochemical experiments were killed 2 h

after injection of LPS or saline with an overdose of sodium

Distinct mechanisms mediate fever and sickness behaviour 4435

ã 2000 Federation of European Neuroscience Societies, European Journal of Neuroscience, 12, 4434±4446

pentobarbital administered i.p. Once the hind paw re¯ex upon plantar

pinching had disappeared, brains were ®xed by intracardiac perfusion

of saline via the ascending aorta followed by 4% paraformaldehyde in

0.1 M borate buffer (pH 9.5 at 10 °C). Brains were post-®xed for 4 h,

and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer

(pH 7.4) for 48 h. Series of 12 frontal 30 mm cryostat sections

through the whole brain were collected in cold cryoproectant (0.05 M

phosphate buffer, 30% sucrose, 30% ethylene glycol) and stored at

±20 °C until immunocytochemical processing.

Immunocytochemistry

Immunocytochemistry was performed as previously described

(Konsman et al., 1999). Commercially available antisera to Fos,

COX-2 and Stat3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA)

diluted, respectively, 1 : 2000, 1 : 500 and 1 : 2000 were used. The

Fos antiserum (cat. number sc-52, lot. J297) was raised against a

synthetic peptide corresponding to amino acids 3±16 at the N-

terminal of human and mouse Fos and did not cross-react with Fos-

related antigens according to the manufacturer. The COX-2 antiserum

(cat. number sc-1747, lot. M19) was raised against a synthetic peptide

corresponding to amino acids 586±604 at the carboxy terminus of the

COX-2 precursor of rat origin. For preabsorption the corresponding

peptide was used following the manufacturer's instructions (sc-

1747P, lot. L288). Stat3 antiserum (cat. number sc-482X, lot. K247)

was generated against a peptide corresponding to amino acids 750±

769 of the carboxy terminus of the mouse and did not cross-react with

Stat1, Stat2, Stat4, Stat5 or Stat6, according to the manufacturer. The

preabsorption test was performed by incubating the antibody with the

synthetic peptide (cat. number sc-485P, lot. B028), as speci®ed by the

manufacturer. A sheep antiserum generated to rrIL-1b (NIBSC) was

used at a ®nal dilution of 1 : 1000.

After washing off the cryoprotectant solution, immunocytochem-

ical processing was performed on a one in four series of free ¯oating

sections using the streptavidin-biotin-immunoperoxidase technique.

Brie¯y, nonspeci®c binding sites were blocked by a 1-h incubation of

sections in Tris-buffered saline (pH 7.4; TBS) containing 0.3% Triton

X-100 and 0.2% casein. The ®rst antibody was added for 60 h at 4 °C

in the same buffer. After four rinses in TBS, sections were treated for

30 min in 0.3% (v/v) hydrogen peroxide, followed by rinsing in TBS.

Sections were incubated for 2 h at room temperature with

biotinylated donkey antisheep/goat or biotinylated donkey antirabbit

immunoglobulins G (Amersham, Les Ulis, France, 1 : 1000) depend-

ing on the ®rst antibody, and stained using the ABC protocol

(Vectastain Elite, Vector Laboratories, Burlingame, CA, USA,

1 : 1000) with nickel-enhanced diaminobenzidine as a chromogene.

Microscopy

The stained sections were examined with a microscope (Leica

Microsystems, Cambridge, UK) and the images were captured by a

high-resolution CCD video camera image and fed into a personal

computer. Subsequently, the Quantimet 600 Image Analysis System

(Leica Microsystems) generated a digitized signal proportional to the

intensity of illumination. Photomicrographs of labelled structures

were obtained using this system and saved as TIFF ®les. Image-

editing software (Adobe Photoshop, Adobe Systems, San Jose, CA,

USA) was used to adjust contrast and brightness only. Image

processing was performed on the grey image by de®ning, for

example, brightness and surface above which labelling has to be

taken into account. Once established, these parameters remained

unchanged. The image was then converted to a binary image and

measurements were taken. The number of Fos-immunoreactive cells

was measured in at least four sections through the ventromedial

preoptic area, the parvocellular part of the paraventricular nucleus of

the hypothalamus, the central nucleus of the amygdala, the

dorsolateral part of the bed nucleus of the stria terminalis and the

nucleus of the solitary tract. These structures were previously shown

to contain Fos-positive cells after i.p. injection of the same dose and

serotype of LPS (Konsman et al., 1999). In the same study, LPS was

shown to induce IL-1b immunoreactivity in circumventricular

organs. To test the hypothesis that vagotomy reduces the induction

of IL-1b in the forebrain after LPS injection, the relative surface of

IL-1b-immunoreactive cells in sections of the subfornical organ

relative to the total surface of this organ was used as a measure of IL-

1b induction. The subfornical organ was chosen among circumven-

tricular organs for its size and regular shape.

Statistical analysis

In order to verify that surgery did not affect behaviour or body

temperature of vagotomized animals compared with sham-operated

animals, duration of social investigation and body temperature were

measured before injection and submitted to a one-way ANOVA. The

duration of social investigation 2 h after injection is expressed as a

percentage of baseline and submitted to a one-way, repeated-measure

ANOVA (surgery as a between-subjects factor and treatment as a

within-subject factor). Temperature data from fever experiments are

presented every 30 min as means 6 SEM and were analysed by a

two-way, repeated-measure ANOVA (surgery and treatment as

between-subject factors and time within-subject factor, respectively).

Post hoc comparisons of individual group means were carried out by

the Newman±Keuls test. Data from immunocytochemistry experi-

ments are expressed as means 6 SEM and were analysed by a two-

way ANOVA (surgery and treatment). In all tests a level of P < 0.05

was considered as statistically signi®cant.

Results

Veri®cation of vagotomy

In animals assigned to fever and behavioural studies, vagotomy

was veri®ed by blockade of the anorexic effect of i.p. injected

cholecystokinin. A two-way ANOVA on the amount of food intake

over 30 min after 18 h of food deprivation revealed a signi®cant

effect of cholecystokinin treatment (F1,17 = 15.1; P < 0.01), sur-

gery (F1,17 = 14.6; P < 0.01) and a signi®cant interaction between

cholecystokinin treatment and surgery (F1,17 = 27.56; P < 0.001).

Cholecystokinin (4 mg/kg, i.p.) signi®cantly inhibited food intake

in sham-operated animals (P < 0.001), but had no effect on food

consumption of vagotomized animals (P > 0.10), as previously

shown (Hansen & Krueger, 1997). As abdominal vagotomy blocks

the satiety effect of 4 mg/kg of cholecystokinin injected intraper-

itoneally (Smith et al., 1981), this indicates that the animals used

in the fever and behavioural studies were successfully vagoto-

mized.

Vagotomy was veri®ed by isolectin I B4 binding in the NTS of

those animals used in immunocytochemical studies. Isolectin I B4

binds vagal afferents in the NTS (Li et al., 1997) and its

distribution in the NTS of sham-operated rats was similar to that

reported previously. High isolectin I B4 binding was found in the

medial and commissural nuclei, as well as in the tractus solitarius

of sham-operated animals, as shown in Fig. 1A. After subdiaph-

ragmatic vagotomy, isolectin I B4 binding in these structures was

highly reduced in most animals (Fig. 1C and D). In two animals,

VGX 12, shown in Fig. 1B, and VGX 24, however, isolectin I B4

binding was intermediate between sham-operated animals and the

4436 J. P. Konsman et al.


remainder of the rats that underwent vagotomy. These animals

were considered to be incompletely vagotomized and excluded

from further analysis.

Vagotomy blocks i.p. IL-1b- and LPS -induced behaviouraldepression but not fever

Behavioural depression

A one-way ANOVA on the duration of social investigation measured

before injection did not reveal any difference between vagotomized

and sham-operated rats (F1,32 = 2.45; P > 0.10). A one-way, repeated

measures ANOVA on the duration of social interaction 2 h after

injection of rrIL-1b or saline revealed a signi®cant effect of rrIL-1binjection (F1,8 = 16.6; P < 0.01) and a signi®cant interaction between

rrIL-1b injection and surgery (F1,8 = 5.71; P < 0.05). rrIL-1binjection signi®cantly reduced social interaction in sham-operated

animals (P < 0.01), but not in vagotomized animals (Fig. 2A). These

animals were immobile, showed piloerection and curled up to

minimize heat loss (Hart, 1988). Vagotomy signi®cantly attenuated

this effect of rrIL-1b (P < 0.05).

A one-way, repeated-measures ANOVA on social interaction 2 h

after injection of saline or LPS, revealed a signi®cant effect of

surgery (F1,8 = 5.63; P < 0.05), LPS injection (F1,8 = 14.5;

P < 0.01) and a signi®cant interaction between surgery and LPS

injection (F1,8 = 9.21; P < 0.05). LPS injection signi®cantly reduced

social interaction in sham-operated animals (P < 0.01), but not in

vagotomized animals (Fig. 2B). Vagotomy signi®cantly attenuated

this effect of LPS (P < 0.01).

Fever

A one-way ANOVA on basal temperatures was measured over the

second hour after light onset revealed that body temperature was

slightly, but consistently lower in vagotomized animals (mean

36.64 6 0.28 °C) compared with sham-operated rats (mean

36.89 6 0.21 °C; F1,68 = 17.0; P < 0.001). This is in accordance

with earlier studies conducted at an ambient temperature of 22 °C (Lin

& Chern, 1985; Watkins et al., 1995; Fleshner et al., 1998) and may be

due to sectioning of thermosensitive afferent ®bres in the hepatic

branch of the vagus nerve (Adachi, 1984). However, in contrast to an

earlier study (Watkins et al., 1995), vagotomy did not prevent the rise

in body temperature after handling stress associated with the i.p.

injection. This indicates that temperature control mechanisms were

largely intact in the present study. This initial rise in temperature

normally persisted for 1 h, after which it returned to basal levels.

A two-way, repeated measures ANOVA revealed a signi®cant effect

of treatment between subjects (F2,28 = 10.9; P < 0.001) and a

signi®cant effect of time within subjects (F13,364 = 12.5; P < 0.001)

as well as a signi®cant interaction between treatment and time

(F26,364 = 7.21; P < 0.001). Both i.p. injection of rrIL-1b and LPS

induced robust fevers, compared with saline injection (P < 0.001 and

P < 0.01, respectively). Body temperature started to rise 2 h after

rrIL-1b or LPS injection (P < 0.01), and was still elevated at the end

of the experiment (P < 0.001) (Fig. 3). Apart from the early stress-

related transient rise, there were no differences in febrile responses to

rrIL-1b and LPS between vagotomized and sham-operated animals

(P > 0.10).

Effects of vagotomy on i.p. LPS-induced Fos expression in theforebrain

As 25 mg/kg of rrIL-1b and 250 mg/kg of LPS gave rise to similar

febrile responses and behavioural depression, immunocytochemical

studies were not performed for rrIL-1b-injected rats. In addition, it

has previously been shown that IL-1b and LPS induce Fos expression

in the same brain structures (Ericsson et al., 1994; Elmquist et al.,

1996).

A two-way ANOVA on the number of Fos-positive cells in the

paraventricular nucleus of the hypothalamus (PVN) revealed a

signi®cant effect of treatment (saline or LPS) (F1,29 = 12.1;

P < 0.01), surgery (sham or vagotomy) (F1,29 = 10.1; P < 0.01)

and a signi®cant interaction between treatment and surgery

(F1,29 = 8.64; P < 0.01). LPS signi®cantly increased the number of

Fos-positive cells in the PVN of sham-operated animals (P < 0.001).

The PVN of LPS-injected, sham-operated animals showed signi®-

cantly more Fos-immunoreactive cells (P < 0.001) (Fig. 4A) com-

pared with LPS-treated vagotomized animals in which no signi®cant

Fos induction was found compared with saline-injected rats

(P > 0.10). A two-way ANOVA on the number of Fos-immunoreactive

cells in the ventromedial preoptic area (VMPO) revealed a signi®cant

effect of treatment and a signi®cant interaction between treatment and

surgery (F1,29 = 10.7; P < 0.01 and (F1,29 = 4.80; P < 0.05), respect-

ively). The VMPO of LPS-treated sham-operated animals contained

signi®cantly greater numbers of Fos-immunoreactive cells compared

with saline injection (P < 0.01). This was not the case in animals that

underwent vagotomy (P > 0.10). As a result, Fos expression was

signi®cantly higher in sham-operated rats treated with LPS compared

with vagotomized animals (P < 0.01, Fig. 4B).

A two-way ANOVA on the number of Fos-positive nuclei in the

central amygdala (Fig. 4C) and dorsolateral bed nucleus of the stria

terminalis (BNST; Fig. 4D) revealed a signi®cant effect of surgery

and a signi®cant interaction between treatment and surgery (central

FIG. 1. Isolectin I B4 binding to vagal ®bres in the nucleus of the solitarytract (NTS) as a means to verify the extent of subdiaphragmatic vagotomy.(A) Isolectin binding in the commissural and medial NTS of a sham-operated animal (Sham 48). (B) NTS sections of an animal that underwentsubdiaphragmatic vagotomy (VGX 12), but still shows considerableisolectin binding compared to the vagotomized animals shown in C and D(VGX 32 and VGX 30, respectively). Animal VGX 12 was therefore judgedto be incompletely vagotomized. Note also the high isolectin binding in thearea postrema (AP) in A and B compared with C and D (cc, central canal).Scale bar, 100 mm.



amygdala; F1,28 = 4.78; P < 0.05 and F1,28 = 5.17; P < 0.05, bed

nucleus; F1,29 = 8.05; P < 0.01 and F1,29 = 7.89; P < 0.01). LPS

injection signi®cantly increased Fos immunoreactivity in the central

amygdala of rats that underwent sham surgery (P < 0.01), and this

increase was blocked by abdominal vagotomy (P < 0.01) (Fig. 4C).

In the dorsolateral bed nucleus of the stria terminalis of sham-

operated rats, LPS administration signi®cantly increased the number

of Fos-positive cells (P < 0.01), an effect not occurring in

vagotomized animals (P > 0.10). Fos expression was signi®cantly

higher in the BNST of LPS-injected, sham-operated animals com-

pared with vagotomized rats. (P < 0.001) (Fig. 4D).

Photomicrographs of Fos expression in VMPO, PVN and central

amygdala of sham-operated and vagotomized animals are shown in

Fig. 5. The pattern of LPS-induced cellular activation in these

structures of sham-operated animals (Fig. 5A, E and I) was blocked

in animals that underwent vagotomy (Fig. 5C, G and K). However,

only complete subdiaphragmatic vagotomy resulted in blockage of

Fos expression, since animals in which isolectin-positive ®bres were

still clearly present at the level of the medial and commissural NTS

(Fig. 1B), showed robust Fos induction after LPS (Fig. 5B, F and J).

Despite a dramatic decrease in LPS-induced Fos expression in the

PVN, some Fos-positive cells were still observed in the dorsomedial

and ventromedial parvocellular part (Fig. 5G). LPS-induced Fos

expression in the organum vasculosum of the laminae terminalis

(OVLT) was not affected by vagotomy (Fig. 6).

LPS-induced COX-2 immunoreactivity

Western blot analysis was performed to examine the speci®city of

the COX-2 antibody. In samples of the hippocampus that is

known to express COX-2 constitutively (Breder et al., 1995), the

COX-2 antibody recognized two bands of »70 kDa (results not

shown), which is consistent with the molecular weights of the two

COX-2 subunits (71 and 73 kDa) described previously (Pomerantz

et al., 1993). After LPS injection COX-2-immunoreactive cells

were found around blood vessels throughout the brain, as

described in earlier studies (Matsumura et al., 1998). This

labelling pattern was not in¯uenced by vagotomy: Fig. 7 shows

COX-2-immunoreactive cells in the meningal strand penetrating

into the OVLT (A) and associated with blood vessels of the

VMPO (B) 2 h after LPS injection into a vagotomized animal.

(C) Saline injection did not induce COX-2-immunoreactive cells

around blood vessels of vagotomized animals. The staining of

neuronal processes in the preoptic area (Fig. 7B, upper right

corner, and Fig. 7C) is in accordance with earlier studies (Breder

et al., 1995; Elmquist et al., 1997a). COX-2 immunoreactivity

was abolished when staining sections that were incubated with the

preabsorbed antibody (D).

LPS-induced changes in Stat3 expression

A previous study showed that the Stat3 antibody used in this

study recognizes one band in brain homogenates (StroÈmberg et al.,

2000). After LPS injection, many Stat3-immunoreactive nuclei,

indicating translocation, were found in the OVLT of both sham-

operated (Fig. 8A) and vagotomized animals (Fig. 8B). No nuclear

Stat3 labelling was seen in the OVLT of sham-operated animals

after saline injection (Fig. 8C). Only a few Stat3-positive nuclei

were seen in the OVLT of vagotomized animals injected with

saline (Fig. 8D). LPS-induced nuclear Stat3 immunoreactivity was

FIG. 2. Behavioural depression in sham-operated and vagotomized rats 2 h after intraperitoneal injection of recombinant rat interleukin-1b (rrIL-1b; 25 mg) orlipopolysaccharide (LPS; 250 mg/kg). Baseline social interaction with a juvenile introduced during 4 min into the cage of the experimental animal wasmeasured just before injection. Social interaction was assessed 2 h later and expressed as percentage of baseline social interaction. Both rrIL-1b and LPSinduce a reduction in social interaction in sham-operated animals. This behavioural depression does not occur in vagotomized rats. sal-Sham, sham-operatedanimals injected with saline; IL-1-Sham, sham-operated animals injected with rrIL-1b; LPS-Sham, sham-operated animals injected with LPS; sal-VGX,vagotomized animals injected with saline; IL-1-VGX, vagotomized animals injected with rrIL-1b; LPS-VGX, vagotomized animals injected with LPS.*P < 0.05; **P < 0.01; ***P < 0.001; LPS-VGX vs. LPS-Sham. *P < 0.05; **P < 0.01 IL-1-VGX or LPS-VGX vs. IL-1-Sham or LPS-Sham. Each data-point is based on n = 5.



not found in sections incubated with the preabsorbed antibody

(results not shown). Constitutive cytoplasmic Stat3 expression in

the brain was found in the cytoplasm of neurons of the PVN, as

well as in the arcuate nucleus (results not shown), as previously

described (HaÊkansson & Meister, 1998; StroÈmberg et al., 2000).

LPS injection did not induce nuclear labelling in these structures.

Mechanisms underlying the effects of vagotomy

Vagotomy does not affect i.p. LPS-induced IL-1b immunoreactivity in

circumventricular organs

Two hours after i.p. injection of LPS, IL-1b-positive cells were

observed in all circumventricular organs and in the choroid plexus, as

previously described (Konsman et al., 1999). Immunoreactive cells

were polar, with several cellular processes arising from the soma that

often encompass blood vessels. No IL-1b immunoreactivity was seen

in saline-treated, sham-operated rats or vagotomized animals. A two-

way ANOVA on the relative surface covered by IL-1b-immunoreactive

cells in the subfornical organ revealed a signi®cant effect of treatment

(F1,27 = 7.18; P < 0.05) (Fig. 9A). No signi®cant differences in IL-

1b immunoreactivity occurred between LPS-treated, sham-operated

rats and vagotomized rats (P > 0.10) (Fig. 9A), indicating that

vagotomy did not affect IL-1b induction after LPS.

Vagotomy blocks LPS-induced Fos expression in the NTS

A two-way ANOVA on the number of Fos-positive nuclei in the NTS

(Fig. 9B) revealed a signi®cant effect of treatment (F1,28 = 6.83;

FIG. 3. Changes in abdominal temperature in sham-operated and vagotomized animals after intraperitoneal injection of recombinant rat interleukin-1b (rrIL-1b; 25 mg; upper graph) or lipopolysaccharide (LPS; 250 mg/kg; lower graph). Temperature starts to rise 2 h after injection of rrIL-1b or LPS and remainselevated until the end of the experiment 5 h later. Temperature responses to rrIL-1b or LPS do not differ between sham-operated and vagotomized animals atany time-point. Abbreviations see Fig. 2. Each data-point is based on n = 5±6.



P < 0.05) and a signi®cant interaction between treatment and surgery

(F1,28 = 8.02; P < 0.01). Subdiaphragmatic vagotomy induced Fos

expression in the NTS at the level of the area postrema in saline-treated

animals, as previously observed (Traub et al., 1996). However, the

effect of vagotomy on the number of Fos-immunoreactive cells in the

NTS of saline-treated rats was not signi®cantly different from animals

that underwent sham surgery (P > 0.10). In contrast, LPS induced a

signi®cant increase in the number of Fos-positive cells in the NTS of

sham-operated animals (P < 0.01), and this increase was prevented by

abdominal vagotomy (P < 0.01) (Fig. 9B).

Discussion

The present ®ndings demonstrate that subdiaphragmatic vagotomy

attenuated behavioural depression, but not fever in response to rrIL-

1b or LPS. In parallel, vagotomy blocked LPS-induced Fos

FIG. 4. Subdiaphragmatic vagotomy prevents the increase in the number of Fos-immunoreactive cells in (A) the paraventricular hypothalamus, (B) ventromedialpreoptic area, (C) central amygdala and (D) bed nucleus of the stria terminalis 2 h after intraperitoneal injection of 250 mg/kg LPS. For abbreviations see Fig. 1.*P < 0.05; **P < 0.01, ***P < 0.001; LPS-VGX vs. LPS-Sham. sal-Sham: n = 10; sal-VGX: n = 11; LPS-Sham: n = 10; VGX-LPS: n = 7.



expression in limbic structures, the PVN and VMPO, but not in the

OVLT. These effects were not due to an attenuation of IL-1b

induction in the brain, but associated with the prevention of Fos

expression in the NTS. Vagotomy did not alter Stat3 translocation in

the OVLT nor induction of the prostaglandin-producing enzyme

COX-2 around blood vessels after LPS injection, suggesting that

circulating IL-6 or prostaglandins acted in the preoptic area to induce

fever.

The observation that vagotomy attenuated rrIL-1b-induced beha-

vioural depression, as measured by reduced social interaction,

con®rms our earlier ®ndings using recombinant human IL-1b and

LPS (BlutheÂ et al., 1996b). Compared with recombinant human IL-

1b, higher doses of rrIL-1b were needed to induce behavioural

depression. Similar doses of this rrIL-1b preparation were also

needed in naive animals (Anforth et al., 1998), suggesting that this

preparation is less active compared with recombinant human IL-1b.

In the same study it was shown that fever readily occurs at lower

doses of rrIL-1b (Anforth et al., 1998). Similarly, lower doses of the

used serotype of LPS induce fever (Horan et al., 1989), but the dose

of LPS used in the present study was the minimal dose needed to

induce behavioural depression (BlutheÂ et al., 1992). The behaviou-

rally active doses of rrIL-1b and LPS resulted in long-lasting fevers,

FIG. 5. Fos expression in the ventromedial preoptic area (VMPO; A±D), paraventricular nucleus of the hypothalamus (PVN; E±H) and central amygdala(CEA; I±L) 2 h after intraperitoneal injection of saline or LPS in vagotomized (VGX) and sham-operated rats. The left lane shows sections of a sham-operated animal (Sham 48) injected with 250 mg/kg LPS. The middle left lane displays sections from an animal (VGX 12) injected with LPS, that was judgedto be incompletely vagotomized based on the persistence of isolectin binding in nucleus of the solitary tract (Fig. 1B). The pattern of Fos expression afterLPS in this animal is similar to that seen in a sham-operated animal (left lane). However, LPS-induced Fos expression is attenuated in a rat that wascompletely vagotomized (VGX 32) (middle right lane). Despite a dramatic decrease in LPS-induced Fos expression in the PVN, Fos-positive cells were stillobserved in the ventromedial parvocellular part (G). The right lane shows a completely vagotomized animal injected with saline (VGX 30). Quanti®ed areasare outlined. 3v, third ventricle; opt, optic nerve. Scale bar, 100 mm.

FIG. 6. Vagotomy does not block LPS-induced Fos expression in theorganum vasculosum of the laminae terminalis. Intraperitoneal injection of250 mg/kg LPS results in a robust Fos expression in the organumvasculosum of the laminae terminalis of (A) vagotomized animals 2 h latercompared with (B) saline injection. oc, optic chiasm. Scale bar, 100 mm.



which were not altered by vagotomy. These ®ndings are consistent

with studies showing that vagotomy attenuates short fevers due to low

doses of IL-1b and LPS, but does not affect fever responses to higher

doses (Hansen & Krueger, 1997; Romanovsky et al., 1997; Caldwell

et al., 1999).

The present experiments allow some further conclusions on the

mechanisms behind the effects of vagotomy on LPS-induced

responses. Although reduced induction of IL-1b expression in the

brain has been proposed to underlie the effects of vagotomy (LayeÂ

et al., 1995; Hansen et al., 1998), IL-1b immunoreactivity in

circumventricular organs was not found to be reduced after LPS in

the present study. A similar ®nding was recently published while this

study was under review (Van Dam et al., 2000). RT-PCR studies

might have revealed a vagus-dependent induction of IL-1b mRNA in

the brain that was not detectable with immunocytochemistry, either

due to a differential regulation of IL-1b mRNA and protein

(Dinarello, 1992) or as a consequence of inferior sensitivity. Our

previous study showed that IL-1b-immunoreactive cells in circum-

ventricular organs are phagocytes (Konsman et al., 1999) that are

known to take up circulating molecules (Murabe et al., 1981). Since

subdiaphragmatic vagotomy does not induce marked differences in

circulating lipopolysaccharide after its i.p. injection (Hansen et al.,

2000), it is unlikely that RT-PCR revealed a vagus-dependent

induction of IL-1b mRNA in circumventricular organs. In addition,

vagotomy does not alter IL-1b mRNA induction in the liver or

peritoneal macrophages (BlutheÂ et al., 1994; Hansen et al., 1998).

This means that IL-1b can still act on IL-1 binding sites in the liver

hilus and associated with vagal paraganglia (Goehler et al., 1997).

Furthermore, IL-1b is known to activate hepatic and gastric vagal

afferent ®bres (Niijima, 1996; Ek et al., 1998). This, together with the

present ®nding that vagotomy prevents LPS-induced Fos expression

in the NTS where vagal afferents terminate, indicates that the effects

of vagotomy are primarily due to interruption of vagal ®bres that are

activated by IL-1b.

The differential effect of vagotomy on behavioural depression and

fever provides evidence for the hypothesis that different cytokine-to-

brain signalling mechanisms are involved. The present study was the

®rst to show that vagotomy prevented LPS-induced Fos expression in

the central amygdala and BNST. This ®nding is consistent with our

hypothesis that the vagus nerve activates limbic structures to produce

behavioural depression. Vagal afferents synapse on catecholaminer-

gic neurons of the NTS (Sumal et al., 1983) that are activated after

peripheral IL-1b or LPS administration (Ericsson et al., 1994; Sagar

et al., 1995). Although the vagus nerve might stimulate catechola-

minergic NTS neurons projecting to the central amygdala (Riche

et al., 1990), local norepinephrine release is not increased after LPS

administration (Lacosta et al., 1999). In fact, the number of central

amygdala projecting neurons in the NTS that are activated after LPS

administration are low compared to the amount of Fos-positive lateral

parabrachial neurons terminating in the central amygdala (Tkacs &

Li, 1999). Since the lateral parabrachial nuclei receive projections

from catecholaminergic neurons in the NTS (Milner et al., 1986), the

vagus nerve is proposed to activate the central amygdala via the

lateral parabrachial nuclei. c-fos mRNA induction in the central

amygdala after IL-1b injection is restricted to encephalinergic

neurons (Day et al., 1999). This, together with the ®nding that

reduction of exploratory behaviour after IL-1b administration in mice

is mediated by opioids (Spadaro & Dunn, 1990), suggests that central

amygdaloid opioid systems play a role in behavioural depression

during sickness.

Vagotomy attenuated Fos induction in the PVN and thus con®rms

earlier studies (Wan et al., 1994; Gaykema et al., 1995). This might

explain inhibition of adrenocorticotropin secretion to i.p. LPS and IL-

FIG. 7. Cyclooxygenase-2 induction in the preoptic area 2 h afterintraperitoneal injection of 250 mg/kg LPS in vagotomized animals. (A)Cyclooxygenase-2 is found in meninges just rostral to the organumvasculosum of the laminae terminalis and (B) around blood vessels in themedial preoptic area after LPS injection. (C) No cyclooxygenase-2 is foundaround blood vessels after injection of saline. (D) Note that some neuronalprocesses were also stained in the preoptic area (B, upper right corner, andC). No staining is found after preabsorbing the antibody with its antigen.3v, third ventricle; oc, optic chiasm. Scale bar, 50 mm.

FIG. 8. Nuclear Stat3 expression indicating translocation in the organumvasculosum of the laminae terminalis of (A) a sham-operated and (B)vagotomized rat 2 h after intraperitoneal injection of 250 mg/kg LPS. No orvery little nuclear labelling is found after saline injection in (C) sham-operated and (D) vagotomized animals. Scale bar, 100 mm.



1b in vagotomized animals (Gaykema et al., 1995; Kapcala et al.,

1996). The NTS sends noradrenergic ®bres to the PVN (Sawchenko

& Swanson, 1982) and local norepinephrine release after i.p. IL-1b is

prevented by vagotomy (Ishizuka et al., 1997). The observation that

LPS-induced Fos expression in the dorsomedial and ventromedial

parvocellular PVN was less affected by vagotomy is possibly due to a

much weaker innervation from the NTS, compared to other parts of

the PVN (Cunningham & Sawchenko, 1988). Interestingly, a small

population of dorsoparvocellular PVN neurons that project to spinal

cord levels innervating brown adipose tissue are speci®cally activated

after intravenous LPS administration, suggesting that they play a role

in fever (Zhang et al., 2000). However, a more detailed analysis is

needed to determine whether this small population is responsible for

fever in i.p. LPS-injected vagotomized animals. Vagotomy also

inhibited Fos expression in the VMPO after LPS. These results seem

dif®cult to reconcile with ®ndings showing that the cyclooxygenase

inhibitor indomethacin attenuates Fos expression in the VMPO and

PVN in response to LPS (Lacroix & Rivest, 1997). These latter

observations provided support for the hypothesis that prostaglandins

produced along blood vessels activate the VMPO and mediate fever

(Elmquist et al., 1997b). However, the present study showed COX-2

around blood vessels, but no Fos-positive cells in the VMPO of

vagotomized animals demonstrating that Fos expression might occur

independently from prostaglandin synthesis around blood vessels.

Nevertheless, prostaglandin E2 is released within the preoptic area

after LPS, and this release is potentiated by norepinephrine (Sehic

et al., 1996) and blocked by vagotomy (Sehic & Blatteis, 1996).

Since the NTS sends noradrenergic projections to the preoptic region

(Saper et al., 1983) and LPS provokes a rise in preoptic

norepinephrine (Linthorst et al., 1995), Fos expression in the

VMPO might be due to local prostaglandin release after activation

of the NTS. COX-2-immunoreactive neurons are found in the

preoptic hypothalamus (Breder et al., 1995), suggesting that

prostaglandin synthesis by these neurons underlies norepinephrine-

induced prostaglandin release in these structures. Since indomethacin

is highly lipophilic and rapidly found in the brain after peripheral

administration (Bannwarth et al., 1990), it is not unlikely that it

attenuated LPS-induced Fos expression (Lacroix & Rivest, 1997) by

blocking vagus-dependent, norepinephrine-induced prostaglandin

synthesis in hypothalamic neurons.

Although short-lasting fevers in response to low doses of LPS

induce fever via the vagus nerve, higher doses are likely to increase

body temperature via circulating mediators (Romanovsky et al.,

1997). Since humoral routes are slower than neural pathways, it is

important to show that their intervention is compatible in time with

fever onset that occurred 2 h after IL-1b or LPS. COX-2 is essential

in fever, since COX-2 knock-out mice do not develop fever in

response to LPS (Li et al., 1999). Two hours after i.p. LPS injection,

COX-2-immunoreactive cells were found in the meningal strand

penetrating into the OVLT and around blood vessels of the preoptic

area. These cells are probably endothelial cells, as shown by

La¯amme et al. (1999) or juxtaposed perivascular microglia

(Elmquist et al., 1997a). Since LPS is detected in plasma within

15 min after its i.p. injection (Lenczowski et al., 1997), it might act

on CD14 receptors present on brain blood vessels (Lacroix et al.,

1998) to induce COX-2. The prostaglandin EP-3 receptor is necessary

for IL-1b- and LPS-induced fever (Ushikubi et al., 1998), and is

found dorsally and laterally from the VMPO (Nakamura et al., 1999).

However, LPS-induced Fos expression was not observed in the

preoptic area in vagotomized animals. Since the EP-3 receptor signals

FIG. 9. Mechanisms underlying the effects of vagotomy. (A) The relative surface occupied by interleukin-1b-positive cells in the subfornical organ 2 h afterintraperitoneal injection of 250 mg/kg LPS does not differ between sham-operated and vagotomized animals. Abbreviations see Fig. 1. sal-Sham: n = 9; sal-VGX: n = 10; LPS-Sham: n = 8; VGX-LPS: n = 9. (B) Subdiaphragmatic vagotomy prevents the induction of Fos in the nucleus of the solitary tract (NTS)2 h after intraperitoneal injection of 250 mg/kg LPS in vagotomized animals. For abbreviations, see Fig. 1. ** P < 0.01, LPS-VGX vs. LPS-Sham. sal-Sham:n = 9; sal-VGX: n = 11; LPS-Sham: n = 10; VGX-LPS: n = 7.



primarily through the inhibitory G-protein (Audoly et al., 1999), and

Fos induction is never observed after inhibitory responses (KovaÂcs,

1998), this might explain the lack of Fos expression in the preoptic

area. Thus, prostaglandins produced by COX-2 around blood vessels

might act on EP-3 receptors in the preoptic area to induce fever

without Fos.

IL-6 is another essential mediator of fever, as IL-6 knock-out

mice do not develop fever after IL-1b or LPS administration

(Chai et al., 1996). Circulating IL-6 rises 1 h after peripheral LPS

administration (Luheshi et al., 1996; Cartmell et al., 2000) and

both peripheral and central pretreatment with anti-IL-6 serum

abolishes fever (Rothwell et al., 1991; Cartmell et al., 2000). We

studied IL-6 action on the brain after LPS injection by detecting

Stat3 that translocates into the nucleus after activation by the

signal transducer gp130 common to the IL-6 receptor family

(Hirano et al., 1994; Heinrich et al., 1998). Two hours after LPS

injection, nuclear Stat3 was found in the OVLT, suggesting that

circulating IL-6 acted on this area devoid of a BBB. Several

®ndings are compatible with the hypothesis that Stat3 and Fos

induction in the OVLT is related to LPS-induced circulating IL-6

action. The OVLT is known to contain mRNA coding for the IL-

6 receptor and the gp130 signal transducer (VallieÁres & Rivest,

1997) and expresses Fos after peripheral IL-6 administration

(VallieÁres et al., 1997). Furthermore, OVLT lesions block LPS-

induced fever in the presence of circulating IL-6 (Caldwell et al.,

1998), suggesting that IL-6 action on the OVLT plays an

important role in fever. When injected into the brain, IL-6

induces fever but not behavioural depression (Lenczowski et al.,

1999). Altogether, this indicates that IL-6 is part of a speci®c

immune-to-brain pathway underlying fever. Although, the relation

between IL-6 action and COX-2 is still unclear, both are essential

in fever (Chai et al., 1996; Li et al., 1999) and were found to

occur at a time-point consistent with the induction of fever in the

present study.

The present work demonstrated that transmission of an immune

message via the vagus nerve activates limbic structures and

induces behavioural depression. Behavioural depression helps to

reduce heat loss upon infection and thus the maintenance of an

elevated body temperature (Hart, 1988). Since doses of rrIL-1band LPS lower than those needed to provoke behavioural

depression readily result in fever, it can be argued that

thermoregulation by behavioural means occurs only during more

severe infections. Although vagotomy may attenuate fever to low

doses of LPS or IL-1b by preventing activation of the VMPO,

our ®ndings show that fever occurring in parallel with behavioural

depression is not affected by vagotomy. In this case, fever is

likely to result from IL-6 action in the OVLT and prostaglandin

synthesis around blood vessels. Taken together, the present

®ndings can be interpreted to suggest that after infection,

increases in the hypothalamic temperature set-point and regulation

of heat loss by behavioural depression are controlled by different

mechanisms of cytokine-to-brain signalling.

Acknowledgements

The authors would like to thank Dr S. Poole (NIBSC, Potters Bar, UK) for hisgift of recombinant rat IL-1b and the corresponding antiserum. These reagentswere produced within the context of the BIOMED I Concerted Action`Cytokines in the Brain' that was funded by the European Community. Wethank J. Keijser (University of Groningen) for the use of and assistance withthe Quantimet 600 Image Analysis System and Professor Dr J.M. Koolhaasand Dr S. LayeÂ for helpful discussion. Supported by INRA, INSERM and theEuropean Community (CT97-0149 and CT97-2492).

Abbreviations

BBB, blood±brain barrier; BNST, bed nucleus of the stria terminalis; COX-2,cyclooxygenase-2; IL-1b, interleukin-1b; IL-6, interleukin-6; i.p., intraper-itoneal; LPS, lipopolysaccharide; NTS, nucleus of the solitary tract; OVLT,organum vasculosum of the laminae terminalis; PVN, paraventricular nucleusof the hypothalamus; rr, recombinant rat; VMPO, ventromedial preoptic area.

References

Adachi, A. (1984) Thermosensitive and osmoreceptive afferent ®bers in thehepatic branch of the vagus nerve. J. Auton. Nerv. Syst., 10, 269±273.

Anforth, H.R., Bluthe, R.M., Bristow, A., Hopkins, S., Lenczowski, M.J.,Luheshi, G., Lundkvist, J., Michaud, B., Mistry, Y., Van Dam, A.M., Zhen,C., Dantzer, R., Poole, S., Rothwell, N.J., Tilders, F.J. & Wollman, E.E.(1998) Biological activity and brain actions of recombinant rat interleukin-1alpha and interleukin-1beta. Eur. Cytokine Netw., 9, 279±288.

Audoly, L.P., Ma, L., Feoktistov, I., de Foe, S.K., Breyer, M.D. & Breyer,R.M. (1999) Prostaglandin E-prostanoid-3 receptor activation of cyclicAMP response element-mediated gene transcription. J. Pharmacol. Exp.Ther., 289, 140±148.

Bannwarth, B., Netter, P., Lapicque, F., PeÂreÂ, P., Thomas, P. & Gaucher, A.(1990) Plasma and cerebrospinal ¯uid concentrations of indomethacin inhumans. Relationship to analgesic activity. Eur. J. Clin. Pharmacol., 38,343±346.

Blatteis, C.M. (1992) Role of the OVLT in the febrile response to circulatingpyrogens. Prog. Brain Res., 91, 409±412.

BlutheÂ, R.M., Dantzer, R. & Kelley, K.W. (1992) Effects of interleukin-1receptor antagonist on the behavioral effects of lipopolysaccharide in rat.Brain Res., 573, 318±320.

BlutheÂ, R.M., Michaud, B., Kelley, K.W. & Dantzer, R. (1996a) Vagotomyattenuates behavioural effects of interleukin-1 injected peripherally but notcentrally. Neuroreport, 7, 1485±1488.

BlutheÂ, R.M., Michaud, B., Kelley, K.W. & Dantzer, R. (1996b) Vagotomyblocks behavioural effects of interleukin-1 injected via the intraperitonealroute but not via other systemic routes. Neuroreport, 7, 2823±2827.

BlutheÂ, R.M., Walter, V., Parnet, P., LayeÂ, S., Lestage, J., Verrier, D., Poole,S., Stenning, B.E., Kelley, K.W. & Dantzer, R. (1994) Lipopolysaccharideinduces sickness behaviour in rats by a vagal mediated mechanism. C. R.Acad. Sci. III, 317, 499±503.

Boulant, J.A. (1981) Hypothalamic mechanisms in thermoregulation. Fed.Proc., 40, 2843±2850.

Breder, C.D., Dewitt, D. & Kraig, R.P. (1995) Characterization of induciblecyclooxygenase in rat brain. J. Comp. Neurol., 355, 296±315.

Caldwell, F.T., Graves, D.B. & Wallace, B.H. (1998) Studies on themechanism of fever after intravenous administration of endotoxin. J.Trauma, 44, 304±312.

Caldwell, F.T.J., Graves, D.B. & Wallace, B.H. (1999) Humoral versus neuralpathways for fever production in rats after administration oflipopolysaccharide. J. Trauma, 47, 120±129.

Cao, C., Matsumura, K., Yamagata, K. & Watanabe, Y. (1996) Endothelialcells of the rat brain vasculature express cyclooxygenase-2 mRNA inresponse to systemic interleukin-1 beta: a possible site of prostaglandinsynthesis responsible for fever. Brain Res., 733, 263±272.

Cartmell, T., Poole, S., Turnbull, A.V., Rothwell, N.J. & Luheshi, G.N. (2000)Circulating interleukin-6 mediates the febrile response to localisedin¯ammation in rats. J. Physiol. (Lond.), 526, 653±661.

Chai, Z., Gatti, S., Toniatti, C., Poli, V. & Bartfai, T. (1996) Interleukin (IL) -6gene expression in the central nervous system is necessary for feverresponse to lipopolysaccharide or IL-1 beta: a study on IL-6-de®cient mice.J. Exp. Med., 183, 311±316.

Cunningham, E.T.J. & Sawchenko, P.E. (1988) Anatomical speci®city ofnoradrenergic inputs to the paraventricular and supraoptic nuclei of the rathypothalamus. J. Comp. Neurol., 274, 60±76.

Dantzer, R. (1994) How do cytokines say hello to the brain? Neural versushumoral mediation. Eur. Cytokine Netw., 5, 271±273.

Day, H.E., Curran, E.J., Watson, S.J. & Akil, H. (1999) Distinctneurochemical poulations in the rat central nucleus and bed nucleus ofthe stria terminalis: evidence for their selective activation by interleukin-1beta. J. Comp. Neurol., 413, 113±128.

Dinarello, C.A. (1992) Dissociation of transcription from translation of humanIL-1 beta: induction of steady state mRNA by adherence or recombinantC5a in the absence of translation. Proc. Soc. Exp. Biol. Med., 200, 228±232.

Ek, M., Kurosawa, M., Lundeberg, T. & Ericsson, A. (1998) Activation of



vagal afferents after intravenous injection of interleukin-1beta: role ofendogenous prostaglandins. J. Neurosci., 18, 9471±9479.

Elmquist, J.K., Breder, C.D., Sherin, J.E., Scammell, T.E., Hickey, W.F.,Dewitt, D. & Saper, C.B. (1997a) Intravenous lipopolysaccharide inducescyclooxygenase 2-like immunoreactivity in rat brain perivascular microgliaand meningeal macrophages. J. Comp. Neurol., 381, 119±129.

Elmquist, J.K., Scammell, T.E., Jacobson, C.D. & Saper, C.B. (1996)Distribution of Fos-like immunoreactivity in the rat brain followingintravenous lipopolysaccharide administration. J. Comp. Neurol., 371, 85±103.

Elmquist, J.K., Scammell, T.E. & Saper, C.B. (1997b) Mechanisms of CNSresponse to systemic immune challenge: the febrile response. TrendsNeurosci., 20, 565±570.

Ericsson, A., KovaÂcs, K.J. & Sawchenko, P.E. (1994) A functional anatomicalanalysis of central pathways subserving the effects of interleukin-1 onstress-related neuroendocrine neurons. J. Neurosci., 14, 897±913.

Fleshner, M., Goehler, L.E., Schwartz, B.A., McGorry, M., Martin, D., Maier,S.F. & Watkins, L.R. (1998) Thermogenic and corticosterone responses tointravenous cytokines (IL-1beta and TNF-alpha) are attenuated bysubdiaphragmatic vagotomy. J. Neuroimmunol., 86, 134±141.

Gaykema, R.P., Dijkstra, I. & Tilders, F.J. (1995) Subdiaphragmatic vagotomysuppresses endotoxin-induced activation of hypothalamic corticotropin-releasing hormone neurons and ACTH secretion. Endocrinology, 136,4717±4720.

Goehler, L.E., Relton, J.K., Dripps, D., Kiechle, R., Tartaglia, N., Maier, S.F.& Watkins, L.R. (1997) Vagal paraganglia bind biotinylated interleukin-1receptor antagonist: a possible mechanism for immune-to-braincommunication. Brain Res. Bull., 43, 357±364.

HaÊkansson, M.L. & Meister, B. (1998) Transcription factor STAT3 in leptintarget neurons of the rat hypothalamus. Neuroendocrinology, 68, 420±427.

Hansen, M.K. & Krueger, J.M. (1997) Subdiaphragmatic vagotomy blocks thesleep- and fever-promoting effects of interleukin-1beta. Am. J. Physiol.,273, R1246±R1253.

Hansen, M.K., Nguyen, K.T., Fleshner, M., Goehler, L.E., Gaykema, R.P.,Maier, S.F. & Watkins, L.R. (2000) Effects of vagotomy on serumendotoxin, cytokines, and corticosterone after intraperitoneallipopolysaccharide. Am. J. Physiol., 278, R331±R336.

Hansen, M.K., Taishi, P., Chen, Z. & Krueger, J.M. (1998) Vagotomy blocksthe induction of interleukin-1beta (IL-1beta) mRNA in the brain of rats inresponse to systemic IL-1beta. J. Neurosci., 18, 2247±2253.

Hart, B.L. (1988) Biological basis of the behavior of sick animals. Neurosci.Biobehav. Rev., 12, 123±137.

Heinrich, P.C., Behrmann, I., MuÈller-Newen, G., Schaper, F. & Graeve, L.(1998) Interleukin-6-type cytokine signalling through the gp130/Jak/STATpathway. Biochem. J., 334, 297±314.

Hirano, T., Matsuda, T. & Nakajima, K. (1994) Signal transduction throughgp130 that is shared among the receptors for the interleukin 6 relatedcytokine subfamily. Stem Cells, 12, 262±277.

Horan, M.A., Little, R.A., Rothwell, N.J. & Strijbos, P.J. (1989) Comparisonof the effects of several endotoxin presss on body temperature andmetabolic rate in the rat. Can. J. Physiol. Pharmacol., 67, 1011±1014.

Ishizuka, Y., Ishida, Y., Kunitake, T., Kato, K., Hanamori, T., Mitsuyama, Y.& Kannan, H. (1997) Effects of area postrema lesion and abdominalvagotomy on interleukin-1 beta-induced norepinephrine release in thehypothalamic paraventricular nucleus region in the rat. Neurosci. Lett., 223,57±60.

Kapcala, L.P., He, J.R., Gao, Y., Pieper, J.O. & DeTolla, L.J. (1996)Subdiaphragmatic vagotomy inhibits intra-abdominal interleukin-1 betastimulation of adrenocorticotropin secretion. Brain Res., 728, 247±254.

Kluger, M.J. (1991) Fever: role of pyrogens and cryogens. Physiol. Rev., 71,93±127.

Konsman, J.P., Kelley, K. & Dantzer, R. (1999) Temporal and spatialrelationships between lipopolysaccharide-induced expression of Fos,interleukin-1beta and inducible nitric oxide synthase in rat brain.Neuroscience, 89, 535±548.

KovaÂcs, K.J. (1998) c-Fos as a transcription factor: a stressful (re) view from afunctional map. Neurochem. Int., 33, 287±297.

Kraly, F.S., Jerome, C. & Smith, G.P. (1986) Speci®c postoperativesyndromes after total and selective vagotomies in the rat. Appetite, 7, 1±17.

Lacosta, S., Merali, Z. & Anisman, H. (1999) Behavioral and neurochemicalconsequences of lipopolysaccharide in mice: anxiogenic-like effects. BrainRes., 818, 291±303.

Lacroix, S., Feinstein, D. & Rivest, S. (1998) The bacterial endotoxinlipopolysaccharide has the ability to target the brain in upregulating its

membrane CD14 receptor within speci®c cellular populations. BrainPathol., 8, 625±640.

Lacroix, S. & Rivest, S. (1997) Functional circuitry in the brain of immune-challenged rats: partial involvement of prostaglandins. J. Comp. Neurol.,387, 307±324.

La¯amme, N., Lacroix, S. & Rivest, S. (1999) An essential role of interleukin-1û in mediating NF-kappa-B activity and COX-2 transcription in cells of theblood±brain barrier in response to a systemic and localized in¯ammationbut not during endotoxemia. J. Neurosci., 19, 10923±10930.

LayeÂ, S., BlutheÂ, R.M., Kent, S., Combe, C., MeÂdina, C., Parnet, P., Kelley, K.& Dantzer, R. (1995) Subdiaphragmatic vagotomy blocks induction of IL-1beta mRNA in mice brain in response to peripheral LPS. Am. J. Physiol.,268, R1327±R1331.

Lenczowski, M.J., BlutheÂ, R.M., Roth, J., Rees, G.S., Rushforth, D.A., vanDam, A.M., Tilders, F.J., Dantzer, R., Rothwell, N.J. & Luheshi, G.N.(1999) Central administration of rat IL-6 induces HPA activation and feverbut not sickness behavior in rats. Am. J. Physiol., 276, R652±R658.

Lenczowski, M.J., Van Dam, A.M., Poole, S., Larrick, J.W. & Tilders, F.J.(1997) Role of circulating endotoxin and interleukin-6 in the ACTH andcorticosterone response to intraperitoneal LPS. Am. J. Physiol., 273,R1870±R1877.

Li, H., Nomura, S. & Mizuno, N. (1997) Binding of the isolectin I-B4from Griffonia simplicifolia to the general visceral afferents in thevagus nerve: a light- and electron-microscope study in the rat. Neurosci.Lett., 222, 53±56.

Li, S., Wang, Y., Matsumura, K., Ballou, L.R., Morham, S.G. & Blatteis, C.M.(1999) The febrile response to lipopolysaccharide is blocked incyclooxygenase-2 (±/±), but not in cyclooxygenase-1 (±/±) mice. BrainRes., 825, 86±94.

Lin, M.T. & Chern, Y.F. (1985) Effects of subdiaphragmatic vagotomy onthermoregulatory responses of rats to different ambient temperatures. Exp.Neurol., 88, 467±470.

Linthorst, A.C., Flachskamm, C., Holsboer, F. & Reul, J.M. (1995)Intraperitoneal administration of bacterial endotoxin enhancesnoradrenergic neurotransmission in the rat preoptic area: relationship withbody temperature and hypothalamic ± pituitary ± adrenocortical axisactivity. Eur. J. Neurosci., 7, 2418±2430.

Luheshi, G., Miller, A.J., Brouwer, S., Dascombe, M.J., Rothwell, N.J. &Hopkins, S.J. (1996) Interleukin-1 receptor antagonist inhibits endotoxinfever and systemic interleukin-6 induction in the rat. Am. J. Physiol., 270,E91±E95.

Matsumura, K., Cao, C., Ozaki, M., Morii, H., Nakadate, K. & Watanabe, Y.(1998) Brain endothelial cells express cyclooxygenase-2 duringlipopolysaccharide-induced fever: light and electron microscopicimmunocytochemical studies. J. Neurosci., 18, 6279±6289.

Milner, T.A., Joh, T.H. & Pickel, V.M. (1986) Tyrosine hydroxylase in the ratparabrachial region: ultrastructural localization and extrinsic sources ofimmunoreactivity. J. Neurosci., 6, 2585±2603.

Murabe, Y., Nishida, K. & Sano, Y. (1981) Cells capable of uptake ofhorseradish peroxidase in some circumventricular organs of the cat and rat.Cell Tissue Research, 219, 85±92.

Nakamura, K., Kaneko, T., Yamashita, Y., Katoh, H., Ichikawa, A. & Negishi,M. (1999) Immunocytochemical localization of prostaglandin EP3 receptorin the rat hypothalamus. Neurosci. Lett., 260, 117±120.

Niijima, A. (1996) The afferent discharges from sensors for interleukin 1 betain the hepatoportal system in the anesthetized rat. J. Auton. Nerv. Syst., 61,287±291.

Opp, M.R. & Toth, L.A. (1998) Somnogenic and pyrogenic effects ofinterleukin-1beta and lipopolysaccharide in intact and vagotomized rats.Life Sci., 62, 923±936.

Pomerantz, K.B., Summers, B. & Hajjar, D.P. (1993) Eicosanoid metabolismin cholesterol-enriched arterial smooth muscle cells. Evidence for reducedposttranscriptional processing of cyclooxygenase I and reducedcyclooxygenase II gene expression. Biochemistry, 32, 13624±13635.

Riche, D., De Pommery, J. & Menetrey, D. (1990) Neuropeptides andcatecholamines in efferent projections of the nuclei of the solitary tract inthe rat. J. Comp. Neurol., 293, 399±424.

Romanovsky, A.A., Kulchitsky, V.A., Simons, C.T. & Sugimoto, N. (1998)Methodology of fever research: why are polyphasic fevers often thought tobe biphasic? Am. J. Physiol., 275, R332±R338.

Romanovsky, A.A., Simons, C.T., SzeÂkely, M. & Kulchitsky, V.A. (1997) Thevagus nerve in the thermoregulatory response to systemic in¯ammation.Am. J. Physiol., 273, R407±R413.

Rothwell, N.J., Busbridge, N.J., Lefeuvre, R.A., Hardwick, A.J., Gauldie, J. &



Hopkins, S.J. (1991) Interleukin-6 is a centrally acting endogenous pyrogenin the rat. Can. J. Physiol. Pharmacol., 69, 1465±1469.

Rothwell, N.J. & Hopkins, S.J. (1995) Cytokines and the nervous system II:Actions and mechanisms of action. Trends Neurosci., 18, 130±136.

Sagar, S.M., Price, K.J., Kasting, N.W. & Sharp, F.R. (1995) Anatomicpatterns of Fos immunostaining in rat brain following systemic endotoxinadministration. Brain Res. Bull., 36, 381±392.

Saper, C.B., Reis, D.J. & Joh, T. (1983) Medullary catecholamine inputs to theanteroventral third ventricular cardiovascular regulatory region in the rat.Neurosci. Lett., 42, 285±291.

Sawchenko, P.E. & Swanson, L.W. (1982) The organization of noradrenergicpathways from the brainstem to the paraventricular and supraoptic nuclei inthe rat. Brain Res., 257, 275±325.

Sehic, E. & Blatteis, C.M. (1996) Blockade of lipopolysaccharide-inducedfever by subdiaphragmatic vagotomy in guinea pigs. Brain Res., 726, 160±166.

Sehic, E., Ungar, A.L. & Blatteis, C.M. (1996) Interaction betweennorepinephrine and prostaglandin E2 in the preoptic area of guinea pigs.Am. J. Physiol., 271, R528±R536.

Smith, G.P., Jerome, C., Cushin, B.J., Eterno, R. & Simansky, K.J. (1981)Abdominal vagotomy blocks the satiety effect of cholecystokinin in the rat.Science, 213, 1036±1037.

Spadaro, F. & Dunn, A.J. (1990) Intracerebroventricular administration ofinterleukin-1 to mice alters investigation of stimuli in a novel environment.Brain Behav. Immun., 4, 308±322.

StroÈmberg, H., Svensson, S.P.S. & Hermanson, O. (2000) Distribution of thetranscription factor Signal Transducer and Activator of Transcription 3 inthe rat central nervous system and dorsal root ganglia. Brain Res., 853, 105±114.

Sumal, K.K., Blessing, W.W., Joh, T.H., Reis, D.J. & Pickel, V.M. (1983)Synaptic interaction of vagal afferents and catecholaminergic neurons in therat nucleus tractus solitarius. Brain Res., 277, 31±40.

Tkacs, N. & Li, J. (1999) Immune stimulation induces Fos expression inbrainstem amygdala afferents. Brain Res. Bull., 48, 223±231.

Traub, R.J., Sengupta, J.N. & Gebhart, G.F. (1996) Differential c-fos

expression in the nucleus of the solitary tract and spinal cord followingnoxious gastric distension in the rat. Neuroscience, 74, 873±884.

Ushikubi, F., Segi, E., Sugimoto, Y., Murata, T., Matsuoka, T., Kobayashi, T.,Hizaki, H., Tuboi, K., Katsuyama, M., Ichikawa, A., Tanaka, T., Yoshida,N. & Narumiya, S. (1998) Impaired febrile response in mice lacking theprostaglandin E receptor subtype EP3. Nature, 395, 281±284.

VallieÁres, L., Lacroix, S. & Rivest, S. (1997) In¯uence of interleukin-6 onneural activity and transcription of the gene encoding corticotrophin-releasing factor in the rat brain: an effect depending upon the route ofadministration. Eur. J. Neurosci., 9, 1461±1472.

VallieÁres, L. & Rivest, S. (1997) Regulation of the genes encoding interleukin-6, its receptor, and gp130 in the rat brain in response to the immuneactivator lipopolysaccharide and the proin¯ammatory cytokine interleukin-1beta. J. Neurochem., 69, 1668±1683.

Van Dam, A.M., Bol, J.G., Gaykema, R.P., Goehler, L.E., Maier, S.F.,Watkins, L.R. & Tilders, F.J. (2000) Vagotomy does not inhibit high doselipopolysaccharide-induced interleukin-1beta immunoreactivity in rat brainand pituitary gland. Neurosci. Lett., 285, 169±172.

Wan, W., Wetmore, L., Sorensen, C.M., Greenberg, A.H. & Nance, D.M.(1994) Neural and biochemical mediators of endotoxin and stress-inducedc-fos expression in the rat brain. Brain Res. Bull., 34, 7±14.

Watkins, L.R., Goehler, L.E., Relton, J.K., Tartaglia, N., Silbert, L., Martin, D.& Maier, S.F. (1995) Blockade of interleukin-1 induced hyperthermia bysubdiaphragmatic vagotomy: evidence for vagal mediation of immune-braincommunication. Neurosci. Lett., 183, 27±31.

Yasui, M., Nakao, A., Yuuki, T., Harada, A., Nonami, T. & Takagi, H. (1995)Immunohistochemical detection of endotoxin in endotoxemic rats. Hepato-Gastroenterology, 42, 683±690.

Zhang, Y., Lu, J., Elmquist, J.K. & Saper, C.B. (2000) Lipopolysaccharideactivates speci®c populations of hypothalamic and brainstem neurons thatproject to the spinal cord. J. Neurosci., 20, 6578±6586.

Zhang, J. & Rivest, S. (1999) Distribution, regulation and colocalization of thegenes encoding the EP2- and EP4-PGE2 receptors in the rat brain andneuronal responses to systemic in¯ammation. Eur. J. Neurosci., 11, 2651±2668.



The vagus nerve mediates behavioural depression, but not fever, in response to peripheral immune...

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